1. Technical Field
The present invention relates to fluorescent-based detection. More particularly, the present invention relates to systems and methods for providing time-gated, time-resolved fluorescent-based detection on an active complementary metal oxide semiconductor (CMOS) biosensor chip.
2. Description of the Related Art
An assay is a qualitative and/or quantitative analysis of an unknown analyte. In one example, an assay can be a procedure that determines the concentration and sequences of DNA in a mixture. In another example, an assay can be an analysis of the type and concentrations of protein in an unknown sample.
Surface-based sensing assays are typically performed in environmental and biomedical diagnostics. The detection of analytes (targets) in a mixture is often implemented at a solid-liquid interface. Passive solid supports, which include glass substrates or polymer membranes, have probe molecules (i.e., “probes”) immobilized on the surface of the solid supports that are used to bind the analytes of interest. Probes include, for example, proteins and nucleic acids. Probes are selected based on the analytes of interest such that there is a strong and specific interaction between a particular type of probe and a particular target.
More than one analyte can be detected using multiplexed detection. In multiplexed detection, different types of probes are arranged in an array on the surface of the solid supports. Each type of probe results in a strong and specific interaction with a different analyte of interest. For example, in DNA analysis, high density microarrays are used to examine gene expressions at the scale of entire genomes by simultaneously assaying mixtures derived from expressed mRNA against thousands of array sites, each bearing probes for a specific gene. Microarrays generally quantify target concentrations in relative terms, for example, in the form of a ratio to hybridization signal obtained using a reference target sample. Other biosensing applications are calibrated to provide absolute target concentrations.
Fluorescent-based detection is commonly used for quantifying the extent of probe-target binding in surface-based sensing assays. In fluorescent-based detection, a target is labeled with a fluorophore molecule, which can cause the target fluorophore to be fluorescent. Traditional microarray scanners include an excitation source, such as a laser, that emits light on the bound target fluorophores. This causes the target fluorophores to emit fluorescent light that is focused and collected (through a generally lossy optical path) onto a cooled charge-coupled device (CCD) or a photomultiplier tube (PMT). Optical filtering is typically used to improve the signal-to-noise ratio (SNR) by removing background light or reflected excitation light. In addition, the arrays are generally sensitive to particular fluorophore concentrations from 108 to 1011 cm−2.
Characteristic lifetimes are associated with each fluorophore. The lifetime is defined by the transient exponential fluorescent decay of the fluorophore once the excitation source is removed. The lifetime, which is typically on the order of nanoseconds for organic dyes, is characteristic of the dye and the environment, and can be used in addition to color and intensity for multiplexed detection. Quantum-dot fluorophores can have lifetimes exceeding 15 nanoseconds at the cost of slightly lower quantum yields. Fluorescent lifetime detection, for example, has been employed for capillary electrophoresis in both the time and frequency domain. Fluorescent lifetime is also sensitive to excited-state reactions, such as fluorescent resonance energy transfer (FRET), which allows for the detection of macromolecular associations labeled by two different fluorophores. For micro-arrays, FRET can be used to detect in situ real-time hybridization kinetics in which both the probe and target are fluorophore-labeled.
In most commercial time-resolved systems, PMT detectors use time-correlated single photon counting (TCSPC). In this case, sensitivity is limited by a dark count, which is typically about 400 Hertz (Hz). For a typical peak quantum efficiency of 25%, this corresponds to a detection limit of approximately 2×105 photons/cm2 sec (i.e., 5×10−7 lux) for an SNR of 20 decibels (dB). For an effective lifetime measurement, a detection limit of at least ten times this can be expected. The time resolution (as determined by the full width at half maximum (FWHM) of the impulse response of the PMT) is limited by jitter in the PMT and instrumentation, and can be less than 50 picoseconds (ρs).
The response of the fluorophore is characterized by the absorption cross-section and quantum yield. Typical fluorophores have cross-sections between 2×10−17 cm2 and 8×10−16 cm2, corresponding to molar extinction coefficients between ε=50,000 cm−1M−1 and 200,000 cm−1M−1. Typical fluorophores also have quantum yields (η) of between 0.05 and 1.0. For example, for η=0.5 and ε=50,000 cm−1M−1, under steady-state illumination, a detection limit of 2×106 photons/cm2 sec could correspond to surface detection limits down to 2×102 molecules/cm2 with an excitation power of 1020 photons/cm2 sec. Detection limits this low are said to characterize single-molecule detection capabilities. Actual detection limits are usually several orders of magnitude greater than this and are limited by background—either the effectiveness of optical filtering in removing the excitation wavelength or in removing stray fluorescence. Lower excitation power also results in higher detection limits.
Known surface-based sensing assays are typically provided on external, macroscopic instruments. Such instruments are often expensive, large, and complex.
Therefore, there is a need in the art to provide a low cost, compact, and integrated chip for surface-based sensing arrays that provides capabilities similar to those on the macroscopic instruments.
Accordingly, it is desirable to provide methods and systems that overcome these and other deficiencies of the prior art.